Neurological Diseases
Alzheimer's Disease (AD) is the most frequent cause of dementia in the United States, affecting over two million individuals each year. It is a degenerative brain disorder characterized clinically by loss of memory, confusion, and gradual physical deterioration. It is the fourth most common cause of death. The etiology of the disease is virtually unknown but has been attributed to various viruses, toxins, heavy metals, as well as genetic defects. The disease is at present incurable.
Until quite recently, AD was thought to account for relatively few of the cases generally classified as senile dementia. Other factors can lead to such a condition, including repetitious mild strokes, thyroid disorders, alcoholism, and deficiencies of certain vitamins, many of which are potentially treatable. It can be appreciated, then, that a diagnostic test specific for AD would be very useful for the clinical diagnosis and proper clinical treatment of subjects presenting with symptoms common to all of these conditions.
The brains of individuals with AD exhibit characteristic pathological accumulations of congophilic fibrous material which occurs as neurofibrillary tangles (NFTs) within neuronal cell bodies, and neuritic (or senile) plaques. NFTs may also be found in the walls of certain cerebral blood vessels. The major organized structural components of NFTs are paired helical filaments (PHFs). Qualitatively indistinguishable amyloid deposits also occur in normal aged brains but in much smaller numbers with restricted topographical distribution.
There has been considerable recent investigative activity regarding the characterization of proteins found in neuritic plaques and NFTs of AD and other neurologic diseases. One of the amyloid proteins initially described by Glenner et al. has been cloned and sequenced (Glenner et al., Biochem. Biophys. Res. Commun. 120:1131-1135 (1984); U.S. Pat. No. 4,666,829). The A4 amyloid protein found in neuritic plaques and blood vessels has been determined to be a component of a 695 amino acid precursor; a protein postulated to function as a glycosylated cell surface receptor (Masters et al., Proc. Natl. Acad. Sci. USA 82:4245-4249 (1985), Kang et al., Nature 325:733-736 (1987)). The gene coding for A4 is located on chromosome 21 (Kang et al., ibid.; Goldgaber et al., Science 235:877-880 (1987); Tanzi et al., Science 235:880-885 (1987); St. George-Hyslop et al., Science 235:885-889 (1987)) but apparently is not linked to the familial form of the disease (Van Broekhoven et al., Nature 329:153-155 (1987)). There appears to be little, if any, protein sequence homology between amyloid A4 and .beta. protein, their higher molecular weight precursor, and nPTP described by the present invention (see discussion below) (Gross et al., J. Clin. Invest. 76:2115-2126 (1985)).
A number of other proteins thought to be associated with the disease have been described, including Ubiquitin, ALZ-50, microtubular-associated proteins .tau. and MAP2, and neurofilament protein (see, for example, Manetto et al., Proc. Natl. Acad. Sci. USA 85:4502-4505 (1988); Wolozin et al., Science 232:648-651 (1986); Selkoe, Neurobiol. Aging 7:425-432 (1986); Perry et al., in: Alterations of the Neuronal Cytoskeleton in Alzheimer's Disease, Plenum, N.Y., pp 137-149 (1987)). More recently, a serine protease inhibitor called .alpha..sub.1 -anti-chymotrypsin has been found in AD amyloid deposits (Abraham et al., Cell 52:487-501 (1988)).
Until this time, there has been no useful diagnostic test for AD. A definitive diagnosis is possible only postmortem, or during life through a brain biopsy, to reveal the presence of the characteristic plaques, tangles, PHFS, and other cerebrovascular deposits which characterize the disorder. Such an invasive surgical procedure is inherently dangerous and is therefore rarely utilized. As a result, the clinical misdiagnosis of AD is estimated to be approximately 20%-30%.
Down Syndrome (DS) results in mental retardation and is associated with a variable constellation of abnormalities caused by trisomy of at least a critical portion of chromosome 21 in some or all cells. No single physical sign is diagnostic and most stigmata are found in some normal persons. In rare patients, no chromosome abnormalities can be detected by routine cytogenetic analysis. Although DS can generally be detected pre- and post-natally by cytogenetic testing, an alternative diagnostic test which measured a parameter other than a gross karyotypic alteration would be useful in identifying and verifying the presence of DS in a subject, either pre- or post-natally.
Neural tube defects refer to defects which develop in the vertebrate embryo in a tube formed from differentiated middorsal ectoderm. In a developing fetus, the neural tube ultimately gives rise to the brain and spinal cord. Thus, defects in the neural tube often result in severe defects in these organs. For example, such defects could include anencephaly, the absence of the cerebral and cerebellar hemispheres of the brain, spina bifida (absence of vertebral arches of the spinal cord through which the spinal membranes (with or without spinal cord tissue) may protrude), meningocele (protrusion of the brain or spinal cord membranes through a defect in the skull or vertebral column), meningomyelocele (protrusion of the membranes and spinal cord through a defect in the vertebral column), or holoprosencephaly (failure of the forebrain to divide into hemispheres).
A simple prenatal diagnostic test, using amniotic fluid, for example, which could detect neural tube defects would be very useful in determining prenatal or early postnatal treatment such as, for example, immediate postnatal surgical intervention.
Pancreatic and other Diseases
Acute pancreatitis or acute pancreatic injury may be caused by multiple factors including alcohol, penetrating peptic ulcer, gallstones, drugs, trauma, uremia, etc. Diffuse abdominal pain, nausea and vomiting, fever, tachycardia, epigastric tenderness and rigidity are cardinal symptoms and physical findings. Often hemoconcentration and intravascular volume depletion are present. Total serum amylase activity of 3-5 times greater than normal has been the diagnostic anchor for such diseases, despite the lack of specificity of this test. Measurement of serum lipase has also been somewhat helpful in this regard. However, serum amylase and lipase may be elevated in this same range in a variety of serious and life-threatening illnesses, some of which are medical emergencies.
For example, it is well-known that serum lipase and total amylase activities may be elevated in perforated ulcer, intestinal obstruction, intestinal infarction, and renal insufficiency. In these conditions, where no pancreatic injury has occurred, the signs and symptoms may be quite similar to those of acute pancreatitis. The treatment of these extrapancreatic causes of elevated amylase and lipase activities, however, is quite different from that for pancreatitis. For example, surgery for acute pancreatitis is discouraged, whereas failure to perform surgery for intestinal infarction can have lethal consequences. Thus, the search for a more specific diagnostic test of acute and chronic pancreatic injury has great clinical significance.
It is therefore clear that a simple, standardized, and relatively inexpensive assay for diagnosing neural tube defects or pancreatic disease, as well as for specifically detecting DS and AD, would be an immensely useful diagnostic tool for the clinician and researcher alike.
Pancreatic Proteins
Pancreatic Thread Protein (PTP) is found in great abundance in the acinar cells of the pancreas and reaches concentrations of 1-2 mg/ml in normal pancreatic fluid as measured by a monoclonal antibody (mAb)-based immunoradiometric assay (M-IRMA) (Gross et al., J. Clin. Invest. 76:2115-2126 (1985)).
PTP in its monomeric form has an apparent molecular weight of approximately 14 kilodaltons (kD), consists of a single polypeptide chain and is rich in aromatic amino acids. The protein has unusual solubility characteristics: it undergoes a pH-dependent fibril formation at pH's between 5.4 and 9.2. The protein forms long "thread like" structures of 7-10 nm (by electron microscopy) when pancreatic fluid is allowed to stand for several hours at 4.degree. C. (Gross et al., J. Clin. Invest. 76:2115-2126 (1985)). Thus, PTP represents one of the major secretory products of the exocrine pancreas in man.
Another pancreatic protein called pancreatic stone protein (PSP) has been described by one research group (DeCaro et al., Biochem. Biophys. Res. Commun. 87:1176-1182 (1979)). Based on amino acid sequence, PSP appears identical to PTP (DeCaro et al., J. Biochem. 168:201-207 (1987)). A similar protein has been identified in bovine pancreas (Gross et al., Proc. Natl. Acad. Sci. USA 82:5627-5631 (1985)).
One group of investigators recently found that treatment of highly pure PHFs with pronase removed a 9.5 kD and 12 kD fragment which included the .tau. microtubular protein (Wischik et al., Proc. Natl. Acad. Sci. USA 85:4506-4510 (1988); Wischik et al., Proc. Natl. Acad. Sci. USA 85:4884-4888 (1988)). The insoluble core protein remaining following pronase digestion had repeating subunits to which a mAb was made. The mAb bound specifically to the core protein but did not bind the .tau. protein (Wischik et al., Proc. Natl. Acad. Sci. USA 85:4506-4510 (1988)). The solubility characteristics and physical appearance (under electron microscopy) of PTP (Gross et al., J. Clin. Invest. 76:2115-2126 (1985)) and the PHF core protein (Wischik et al., Proc. Natl. Acad. Sci. USA 85:4506-4510 (1988); Wischik et al., Proc. Natl. Acad. Sci. USA 85:4884-4888 (1988)) are similar.